EP1624071B1 - Nachweisverfahren für lebenfähige Zellen - Google Patents

Nachweisverfahren für lebenfähige Zellen Download PDF

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EP1624071B1
EP1624071B1 EP05015801A EP05015801A EP1624071B1 EP 1624071 B1 EP1624071 B1 EP 1624071B1 EP 05015801 A EP05015801 A EP 05015801A EP 05015801 A EP05015801 A EP 05015801A EP 1624071 B1 EP1624071 B1 EP 1624071B1
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Prior art keywords
dye
viable cells
fluorescent
sample
fluorescent dye
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French (fr)
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EP1624071A2 (de
EP1624071A3 (de
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Takaaki Fuji Electric Adv.Techn. Co. Ltd Mizutani
Naohiro Fuji Electric Adv.Techn. Co. Ltd Noda
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Fuji Electric Co Ltd
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Fuji Electric Holdings Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a method of detecting viable cells in a sample, in particular, to a method of accurately detecting viable cells, the method facilitating discrimination between a viable cell and an inclusion such as a dust by staining the sample using a fluorescent dye and a quenching dye.
  • Patent Document 1 discloses a method of quantification of live and dead microbes using fluorescent dyes of fluorescein diacetate and propidium iodide.
  • the microbes are doubly stained by these dyes and illuminated by excitation light. Detecting fluorescent light at a specific wavelength emitted from the live cells stained with fluorescein diacetate and fluorescent light at a specific wavelength emitted from the dead cells stained with propidium iodide, the numbers of live and dead cells are quantified based on the numbers of the emitted fluorescent light.
  • Patent Document 2 discloses a method of quantification of microbes to count live and dead cells through two steps and comparison of them.
  • the whole sample of the microbes is stained with a fluorescence reagent that allows only dead cells to emit fluorescent light, and fluorescent dead cells are counted.
  • the whole sample is stained again with the same fluorescent reagent and the fluorescent dead cells are counted. Comparing the numbers counted in the two steps, live and dead cells are quantified.
  • Patent Document 3 discloses a method of measuring a number of viable cells and/or viability ratio. The method measures an intensity of fluorescent light emitted from a sample treated with a nucleic acid fluorescent dye that allows only dead cells to be stained, and measures an intensity of fluorescent light emitted from a sample treated with the nucleic acid fluorescent dye and subjected to a treatment to disrupt the cell membrane. The two intensities are compared to determine the cell number and/or the viability.
  • Patent Document 4 discloses a method of detecting viable cells comprising a process of detection or measurement of a dye or fluorescence in a cell sample having a dye or a fluorescent enzyme substrate added, wherein the detection or the measurement is carried out in presence of an absorber that does not permeate through a cell membrane and absorbs the dye or the light emission from the fluorescent enzyme substrate.
  • the fluorescent enzyme substrate is a compound selected from the group consisting of 5-carboxyfluorescein diacetate acetoxymethyl ester, 5-(6-) carboxyfluorescein diacetate, 2',7'-bis-(2-carboxyethyl)-5-(6-) carboxyfluorescein acetoxymethyl ester, 5-(6-) sulfofluorescein diacetate, fluorescein diacetate, calcein acetoxymethyl ester, 5-chloromethyl fluorescein diacetate, 5-(6-) carboxyfluorescein diacetate succinimidyl ester, and fluorescein-5-carbonylazido diacetate.
  • the dye is a compound selected from the group consisting of acridine orange, bis benzimidofluorochrome trihydrochlorate, 4',6'-diamino-2-phenyl indole, SYTO9, SYTO10, SYTO11, SYTO12, SYTO13, SYTO14, SYTO15, SYTO16, SYTO17, SYTO20, SYTO21, SYTO22, SYTO23, SYTO24, SYTO25, ("SYTOn are manufactured by Invitrogen Corporation), hexidium iodide, and dihydroethidium.
  • the absorber is a compound selected from the group consisting of cytochrome C, hemoglobin, and blue dextran.
  • Patent Document 5 discloses a method of quantifying relative amount of viable cells by staining a sample containing the viable cells with two types of dyes and measuring intensity of fluorescence from the sample.
  • the first of the two types of dyes is a fluorescent dye that is accumulated in viable cells only.
  • the second is a dye that quenches the fluorescence of the fluorescent dye and is permeable to dead cells but excluded by viable cells.
  • the disclosed combination of the dyes include a combination of a fluorescent dye of fluorescein diacetate and a quenching dye of eosin Y, and a combination of a fluorescent dye of calcein-AM and a quenching dye of trypan blue.
  • Non-patent Document 1 discloses a method of quantifying viable cells.
  • the sample is doubly stained using carboxyfluorescein diacetate (CFDA), which is a reagent for viable cells to emit fluorescent light; and hemoglobin, which quenches the fluorescence of CFDA leaked from the viable cells.
  • CFDA carboxyfluorescein diacetate
  • the stained sample is illuminated with an excitation light and the emitted fluorescence from the viable cells stained by the CFDA is detected by a photomultiplier connected to a fluorescent microscope, to determine the number of viable cells from the detected intensity of the fluorescence.
  • CFDA carboxyfluorescein diacetate
  • Heglobin which quenches the fluorescence of CFDA leaked from the viable cells.
  • the stained sample is illuminated with an excitation light and the emitted fluorescence from the viable cells stained by the CFDA is detected by a photomultiplier connected to a fluorescent microscope, to determine the number of viable cells from the detected intensity of the
  • Patent Document 3 Japanese Unexamined Patent Application Publication No. H10-99096
  • Patent Document 4 Japanese Unexamined Patent Application Publication No. 2002-34594
  • Patent Document 5 U.S. Patent No. 6,459,805
  • Nonpatent Document 1 Y. Hansson et al.; Journal of Immunological Methods, 100 (1987), pp. 261-267
  • Patent Document 1 has a drawback in that the fluorescein diacetate is vulnerable to dissociation and stains inclusions other than viable microbes, inhibiting accurate quantification of viable cells in the case of samples containing live and dead microbes and other inclusions together.
  • Patent Document 2 also involves disadvantages. In addition to the need for a complicated process of sterilization, the conditions of the sterilization affect the measured result and must be closely examined.
  • Patent Document 3 also involves problems.
  • the treatment to disrupt the cell membrane is troublesome, and the conditions of the treatment must be closely checked.
  • the objects of the measurement are confined since the cells having a cell wall cannot be measured.
  • the trypan blue is a harmful substance requiring careful handling.
  • Non-patent Document 1 Problems in the methods of Patent Document 4 and Non-patent Document 1 include that the absorber of the emitted light from the dye or fluorescent enzyme substrate is a protein such as cytochrome C, hemoglobin or the like, which needs cold storage and is difficult to maintain stable quality.
  • the blue dextran is expensive.
  • Patent Document 5 involves a problem in fast quantification because relatively long time (about 30 min) is required for staining the sample with the fluorescent dye.
  • the quenching dye of eosin Y does not absorb the fluorescent light of the carboxyfluorescein diacetate in the range of the wavelength between 550 nm to 650 nm, so eosin Y cannot quench the fluorescence of the fluorescein diacetate satisfactorily.
  • an object of the present invention is to provide a method of detecting viable cells, the method detecting and quantifying viable cells in a sample quickly, readily, and accurately, using reagents with safety, stability in storage, and a low cost.
  • a method of detecting viable cells according to the present invention comprises steps of:
  • the sample is doubly stained by a fluorescent dye and a quenching dye (a) that is permeable through a membrane of a viable cell, (b) that does not absorb the fluorescent light of the fluorescent dye at a pH value in the viable cells, and (c) that absorbs the fluorescent light of the fluorescent dye at a pH value substantially different from the pH value in the viable cells, and the sample is kept at a pH value substantially different from the pH value in the viable cells in a method of detecting viable cells of the invention, the fluorescent light coming from the inclusions stained by the fluorescent dye is absorbed and quenched by the quenching dye, eliminating influence of the inclusions. Therefore, the viable cells in the sample are detected and quantified quickly, readily, and accurately.
  • the fluorescent dye is capable of staining mainly viable cells. This aspect of embodiment further eliminates the influence of inclusions.
  • the fluorescent dye is a substance that fluorescently labels nucleic acid or an enzyme substrate that becomes fluorescent on enzymatic degradation. This aspect of embodiment allows viable cells in a sample to be stained quickly and readily.
  • the quenching dye is preferably selected from compounds having conjugated double bond(s) absorbing light with a wavelength of the fluorescent light of the fluorescent dye, and more preferably selected from an aromatic compound having at least two aromatic rings, an aromatic compound having at least one fused aromatic ring, and a compound having an unsaturated hydrocarbon structure.
  • Specific examples of the preferable material of the quenching dye include anthocyanins and the compounds represented by the structural formulas (I) through (IX).
  • each of R 1 through R 11 which may be the same or different, represents a hydrogen atom, a methyl group, an aliphatic chain or an ester of fatty acid of two or more carbon atoms, iodine, or bromine.
  • Me represents iron, copper, or magnesium; each of R 1 through R 7 represents a hydrocarbon group; and each of R 8 through R 12 represents a hydrocarbon group, a hydrogen atom; or a carbon atom.
  • each of R 1 through R 8 represents a hydrocarbon group
  • each of R 9 through R 12 represents a hydrocarbon group, a hydrogen atom, or a carbon atom.
  • each of R 1 through R 4 represents a hydrocarbon group, a hydrogen atom, or a carbon atom, and n is an integer from 1 to 11.
  • quenching dyes effectively quench the inclusions that are non-specifically stained with a fluorescent dye.
  • the quenching dyes are equivalent or superior in safety, storage stability, and cost as compared with traditionally used quenching dyes.
  • the step (2) is conducted using a quenching dye that absorbs the fluorescent light of the fluorescent dye at an alkaline pH, and the step (3) is conducted keeping the sample stained with the fluorescent dye and the quenching dye at an alkaline pH.
  • the step (2) advantageously is conducted using a quenching dye that absorbs the fluorescent light of these fluorescent dye at an acidic pH, and the step (3) is conducted keeping the sample stained with the fluorescent dye and the quenching dye at an acidic pH.
  • step (3) of an aspect of embodiment according to a method of detecting viable cells of the invention it is preferable to collect only light with a wavelength of the fluorescent light of the fluorescent dye and save this fluorescent light as an image.
  • an image of the fluorescent light is saved that is emitted by viable cells stained with the fluorescent dye, while the fluorescent light from the inclusions stained with the fluorescent dye is quenched by the quenching dye, and an image of the fluorescent light is not saved that is emitted by the quenching dye excited by the energy absorbing the fluorescent light of the fluorescent dye. Therefore, only the fluorescent light from the viable cells is detected.
  • the fluorescent light emitted from the sample it is preferable to collect the fluorescent light emitted from the sample and save the collected fluorescent light as an image in color, and to distinguish between the fluorescent light arising from the fluorescent dye and the fluorescent light from other sources.
  • the viable cells are saved as an image of the fluorescent light of the fluorescent dye, and the inclusions are saved as an image of the fluorescent light of the quenching dye.
  • the quenching dye emits fluorescence with longer wavelength than the fluorescent dye.
  • a method of the invention When a method of the invention is applied to measuring a sample containing viable cells and inclusions, in particular to detecting bacteria in food, the interference due to the inclusions that are stained by the fluorescent dye together with the live bacteria can be avoided. Therefore, the detection of the live bacteria is carried out accurately, readily, and in a short time.
  • Cells in the invention are not limited to special types, but include bacteria, such as Escherichia coli, Staphylococcus, Pseudomonas, Bacillus, and Serratia, and fungi such as yeast; and further include animal cells and plant cells.
  • the viable cell means live microbes of the bacteria and fungi and live cells of other organisms.
  • Fluorescent dyes used in the invention can be any material that fluorescently stains cells, without any special limitation.
  • Preferable materials include substances that fluorescently label nucleic acid and enzyme substrates that become fluorescent on enzymatic degradation.
  • the substance that fluorescently labels nucleic acid can be any substance that permeates cell membrane and binds with DNA for example, DAPI (4',6-diamidino-2-phenylindole dihydrochloride) (Reference: Sigma general catalog, p631, 2004-2005 edition).
  • Examples of the enzyme substrate that becomes fluorescent on enzymatic degradation are carboxyfluorescein diacetate (CFDA), Calcein-AM, carboxyfluorescein diacetate succinimidyl esters, CMFDA, PFB-FDA, 5-(and 6-) chloromethyl SNARF-1 acetate, 2'-7'-dichlorodihydrofluoresceindiacetate acetate ester, CM-H 2 DCFDA, carboxyeosin diacetate succinimidyl ester (Reference: "Molecular Probes Handbook of Fluorescent Probes and Research Products (Ninth Edition)", Invitrogen Corporation, (2002)).
  • the enzyme substrates that become fluorescent on enzymatic degradation are particularly favorable for use in the invention because of safety from carcinogenesis and property to stain mainly viable cells.
  • the quenching dye used in the invention is, without any special limitation, any material (a) that permeates a membrane of a viable cell, (b) that does not absorb the fluorescent light of the fluorescent dye at a pH value in the viable cell, and (c) that absorbs the fluorescent light of the fluorescent dye at a pH value substantially different from the pH value in the viable cell.
  • Preferable materials include compounds having a conjugated double bond(s) that absorbs fluorescent light with a wavelength of the fluorescence of the fluorescent dye; more preferably include aromatic compounds having at least two aromatic rings, aromatic compounds having at least one fused aromatic ring, and compounds having an unsaturated hydrocarbon structure.
  • Specific examples of the preferable material include anthocyanins and the compounds represented by the structural formulas (I) through (IX).
  • each of R 1 through R 11 which may be the same or different, represents a hydrogen atom, a methyl group, an aliphatic chain or an ester of fatty acid of two or more carbon atoms, iodine, or bromine.
  • Me represents iron, copper, or magnesium; each of R 1 through R 7 represents a hydrocarbon group; and each of R 8 through R 12 represents a hydrocarbon group, a hydrogen atom, or a carbon atom.
  • each of R 1 through R 8 represents a hydrocarbon group
  • each of R 9 through R 12 represents a hydrocarbon group, a hydrogen atom, or a carbon atom.
  • each of R 1 through R 4 represents a hydrocarbon group, a hydrogen atom, or a carbon atom, and n is an integer from 1 to 11.
  • Examples of the compounds represented by the structural formula (I) include 3', 3", 5', 5"-tetraiodophenol-sulfonphthalein, Phenol Red, Cresol Red, m-Cresol Purple, Thymol Blue, Bromothymol Blue, and p-Xylenol Blue.
  • Examples of the compounds represented by the structural formula (II) include o-Cresolphthalein and Phenolphthalein.
  • Examples of the compounds represented by the structural formula (III) include Aurin.
  • Examples of the compounds represented by the structural formula (IV) include p-Naphtholbenzein.
  • Examples of the compounds represented by the structural formula (V) include 3',3",5',5"-tetraiodophenol-sulfonphthalein sodium salt, Phenol Red sodium salt, Cresol Red sodium salt, m-Cresol Purple sodium salt, Thymol Blue sodium salt, Bromothymol Blue sodium salt, and p-Xylenol Blue sodium salt.
  • Examples of the compounds represented by the structural formula (VI) include ⁇ -Naphtholphthalein.
  • Examples of the compounds represented by the structural formula (VII) and the structural formula (VIII) include porphyrin compounds, specifically, protohem, protochlorophyl, and protoporphyrin.
  • Examples of the compounds represented by the structural formula (IX) include compounds having an unsaturated hydrocarbon structure, specifically, ß-carotene and Curcumin. Examples of the compounds represented by the structural formula (IX) further include aromatic compounds having at least two aromatic rings, specifically, Curcumin and bis(2,4-dinitrophenyl) acetic acid ethyl ester.
  • anthocyanins examples include plant-originated dyes, specifically, pelargonin, calystephin, fragarin, cyanin, chrysanthemin, shisonin, keracyanin, delphinine, nasunin, hyacin, and enin.
  • the pH value in viable cells is generally in the range of 6.8 to 7.4 (Reference: "Molecular Probes Handbook of Fluorescent Probes and Research Products (Ninth Edition)", Invitrogen Corporation, p829, (2000)).
  • the quenching dyes scarcely absorb the fluorescence of the fluorescent dyes at the pH value in viable cells, but absorb the fluorescence of the fluorescent dyes at more slightly acidic(about pH5-pH6.8) and more mildly alkaline(about pH7.4-pH9) than that of the pH value in viable cells.
  • the quenching dyes are used in an appropriate selection corresponding to the fluorescent dye used together. In case the fluorescent dye emits green fluorescence like CFDA or emits blue fluorescence like DAPI, the quenching dye is preferably Phenol Red and/or Cresol Red, for example.
  • the fluorescence of the fluorescent dyes emitted from inclusions are absorbed and quenched, in the present invention, by such a quenching dye, absorption wavelength range of which overlaps with the wavelength range of the fluorescence of the fluorescent dyes.
  • a quenching dye quenches a fluorescent dye in the method of invention.
  • the resonance energy transfer may occur between two functional groups in certain substance having in one molecule a functional group that absorbs electromagnetic energy and another functional group that emits fluorescent light. It is further known that the resonance energy transfer may occur between two molecules each individually having such functional group exhibiting respective property as described above (Reference: " Gendai Kagaku” (in Japanese), Tokyo Kagaku Dozin Co., Ltd. p22 (2002 )).
  • n-component of a double bond involves for a functional group to absorb electromagnetic energy in the visible to ultraviolet region.
  • a specific example of such a functional group is an aromatic ring having a conjugated double bond.
  • the gap between electronic states is narrower and the wavelength of the absorption band of electromagnetic energy in the visible to ultraviolet region shifts to longer wavelength region, (Reference: a translation into Japanese from " Barlow: Physical Chemistry for Bioscience, Second Edition", p300-312, Tokyo Kagaku Dojin, 1983 ).
  • This type of aromatic molecule receives or loses one or several hydrogen ions, to change its electric charge.
  • the change of electric charge is observed in the wavelength of the absorption band of electromagnetic energy in the visible to ultraviolet region.
  • the molecule binds more tightly with electrons and the absorption band of electromagnetic energy shifts towards shorter wavelength region.
  • the positive charge decreases or the negative charge increases, the absorption band shifts towards longer wavelength region.
  • the quenching dye described above causes wavelength shift of the absorption band of electromagnetic energy corresponding to the difference of pH value.
  • the pH value inside viable cells is retained approximately constant mitigating influence of the pH outside the cells, (Reference: a translation into Japanese of "J. G. Black: Microbiology - Principles and Explorations", p152, Maruzen Co. Ltd., 2003,) and the viable cells emit fluorescence of the fluorescent dye.
  • the inclusions other than the viable cells readily influenced by the environmental pH and the quenching dye absorbs and quenches the fluorescence of the fluorescent dye (actually, emits fluorescent light at a wavelength longer than that of the fluorescence of the fluorescent dye).
  • the viable cells can be discriminated from the inclusions other than the viable cells.
  • Fig. 1 shows spectral characteristics of excitation wavelength and fluorescence wavelength of CFDA, and spectral characteristics of absorption wavelength of Phenol Red and Cresol Red under pH of 8.6.
  • the CFDA emits fluorescent light in the range of 480 nm to 650 nm on illumination by light at 470 nm. Since the absorption wavelength of Phenol Red overlaps the fluorescence wavelength of the CFDA, the Phenol Red absorbs and quenches the fluorescence of CFDA and emits red light in the range of 550 nm to 800 nm.
  • Step of fluorescently staining cells by adding a fluorescent dye to a sample or bringing a sample in contact with a fluorescent dye
  • the cells in a sample can be stained with a fluorescent dye after fixing onto a filter or an adhesive sheet as shown in (i) to (iii) below in the method of the invention, although the cells can be stained by adding a fluorescent dye solution directly to the sample.
  • sample are supplied for measurement preferably after a pre-treatment such as degreasing, protein removal, filtration, or centrifugation.
  • a sample not in a liquid state is preferably used after extracting the cells into a liquid using a breaking and dispersing apparatus such as a mixer or a stomacher.
  • fluorescence staining can be conducted on the cells as trapped on the filter in step (i)
  • the fluorescence staining can also be conducted after adhering an adhesive sheet to the entire surface of the filter and transferring the cells trapped on the filter onto the adhesion layer of the adhesive sheet.
  • a usable adhesion sheet can have a structure comprising an adhesion layer on a base material, the adhesion layer exhibiting sufficient adhesivity to capture the cells trapped on the filter and having a smooth surface structure.
  • the adhesion layer only needs sufficient adhesivity to capture the cells trapped on the filter and is not imposed with any special limitation.
  • the adhesion layer is not impregnated with the fluorescent dye for staining the cells and the captured cells scarcely move in the melted adhesion layer.
  • the adhesion layer is preferably composed of an adhesive that is insoluble in water for example, acrylic adhesives, rubber adhesives, or silicone adhesives.
  • the acrylic adhesive can be copolymer composed of at least one principal component of alkyl methacrylate ester and at least one copolymerizing monomer of hydrophilic monomer.
  • the alkyl methacrylate can be selected from ethyl methacrylate, propyl methacrylate, butyl methacrylate, hexyl methacrylate, octyl methacrylate, nonyl methacrylate, decyl methacrylate, and the like.
  • the hydrophilic monomer can be selected from methacrylic acid, itaconic acid, maleic acid, hydroxyethyl methacrylate, methoxyethyl methacrylate, ethoxyethyl methacrylate, butoxyethyl methacrylate, ethylene glycol methacrylate, and the like.
  • the adhesion layer composed of such an adhesive is preferably treated for improving adhesion performance using a thermal crosslinking agent such as an isocyanate compound, an organic peroxide, an epoxy group-containing compound, a metal chelate compound, and the like.
  • the adhesion layer is also preferably subjected to a treatment for improving rigidity by irradiating ultraviolet light, ⁇ ray, or electron beam to crosslink the adhesive.
  • the rubber adhesive can be composed of a principal polymer selected from natural rubber, polyisobutylene, polyisoprene, polybutene, styrene-isoprene block copolymer, and styrene-butadiene block copolymer, and an additive resin for providing adhesivity selected from rosin, terpene, chroman-indene resin, terpene-phenol resin, and petroleum resin.
  • the silicone adhesive can be an adhesive mainly composed of dimethyl polysiloxan, for example.
  • Acrylic adhesives and silicone adhesives being highly transparent, are particularly favorable in the method of invention in view of little influence on the optical performance in obtaining fluorescent image.
  • the thickness of the adhesion layer is preferably in the range of 5 to 100 ⁇ m in view of adhesivity and adaptability to the filter and ability to capture the cells.
  • the surface of the adhesion layer preferably has a smoothness (irregularity) of at most 20 ⁇ m. The smoothness can be determined by observing the cross-section of the adhesive sheet using a surface roughness probe or an electron microscope and measuring the average height from a bottom of dent to a top of protrusion on the surface of the adhesive material.
  • the base material of the adhesion sheet only needs not to form large irregularity on the surface of the adhesion layer and to be flexible so as to be contact-bonded to any curved surface and narrow place on the surface, without any special limitation.
  • Preferable materials include polyester, polyethylene, polyurethane, poly (vinyl chloride), woven fabric, nonwoven fabric, paper, and polyethylene laminated paper, among which particularly favorable are polyester, polyethylene, poly (vinyl chloride), and polyurethane because of their high smoothness.
  • the thickness of the base material only needs enough strength as a supporting body and preferably in the range of 5 to 200 ⁇ m.
  • the adhesion sheet can be manufactured by forming an adhesion layer from the adhesive exemplified above on the substrate by a known method and used by cutting to a desired shape.
  • a process of fluorescence staining is conducted on the cells trapped on a filter in the process (i) above, or the cells transferred onto an adhesion sheet in the process (ii) above.
  • a fluorescent dye solution is used that is prepared by dissolving the CFDA in a buffer solution (pH value of 6 to 9; preferably a phosphate buffer saline of pH value in the range of 7.6 to 8.6) with a CFDA concentration in the range of 300 to 3,000 ⁇ g/ml.
  • a fluorescent dye solution is used that is prepared by dissolving the DAPI in a buffer solution (pH value in the range of 5 to 9; preferably a phosphate buffered saline of pH value of in the range of 6 to 8) with a DAPI concentration in the range of 0.1 to 10 mg/ml, more preferably about 1 mg/ml. If the concentration of the fluorescent dye is too low, the cells can not be stained sufficiently, while if the concentration of the fluorescent dye is too high, the inclusions such as dust are stained excessively and quenching by a quenching dye becomes insufficient.
  • the fluorescent dye solution is filtered through a filter with perforation of 0.2 ⁇ m to avoid contamination with disturbing microbes.
  • an antiseptic such as sodium azide can be contained as necessary.
  • the sodium azide for example, is added in an amount of final concentration of 0.1 to 5 mg/ml.
  • a fluorescent dye solution is dropped on a filtration surface of the filter or on an adhesion layer (surface of capturing microbes) of the adhesion sheet, or alternatively, the filter or the adhesion sheet is dipped in a fluorescent dye solution, and left at 2 to 40°C for 30 sec to 3 min and then excessive fluorescent dye solution is rinsed away with a cleaning liquid.
  • the cleaning liquid is preferably a buffer solution at a pH value fitting to coloring of the fluorescent dye used.
  • a buffer solution is a phosphate buffered saline at preferably a pH value of 6 to 9, more preferably pH of 7.6 to 8.6.
  • the buffer solution is used after filtration through a filter with perforation of 0.2 ⁇ m.
  • the quenching dye is used after dissolving in a buffer solution at an appropriate pH value fitting to the dye.
  • a proper pH is a somewhat more acidic pH, i.e. about pH5-pH6.8, than a pH inside of the viable cells and a somewhat more alkaline pH, i.e. about pH7.4-pH9, than a pH inside of the viable cells, and substantially different from the pH in viable cells.
  • a quenching dye solution at a pH value in a range of strongly acidic or strongly alkaline is not appropriate because such a quenching dye solution may not be able to absorb and quench the fluorescence of the fluorescent dye satisfactorily and may kill the viable cells.
  • a quenching dye solution is used that is prepared by dissolving the Phenol Red in a buffer solution (preferably at the pH value of 8 to 9; more preferably a phosphate buffered saline of a pH value of about 8.6) with a Phenol Red concentration preferably in the range of 1 to 30 mg/mL, more preferably in the range of 1 to 10 mg/mL.
  • Fig. 6 shows absorption spectral characteristics of Phenol Red whose concentration is 0.01mg/mL at each pH.
  • absorption near wave length 550nm is lower, maximum absorption wave length change from a wave length near 550nm to that of near 440nm at a pH lower than pH7. It can distinguish between the viable cells and inclusions when there is a difference between absorption intensities of the quenching dye corresponding to a pH inside of the viable cells and a pH outside of the viable cells.
  • the process of staining is conducted on a condition of a pH in which a difference occurs in an absorption intensity at a wavelength of near 550nm.
  • Phenol Red is used for a quenching dye
  • a pH of the quenching dye is preferably pH8-pH9, more preferably pH8.6. In Fig.
  • a quenching dye solution is used that is prepared by dissolving the Cresol Red in the buffer solution same as above with a Cresol Red concentration of preferably in the range of 0.1 to 2.5 mg/mL, more preferably in the range of 1 to 2.5 mg/mL.
  • the concentration of the quenching dye is too low, the fluorescence of the fluorescent dye coming from inclusions cannot be satisfactorily quenched and the detection error increases. If the concentration of the quenching dye is too high, in the case of Phenol Red, the fluorescence from the viable cells becomes feeble, and in the case of Cresol Red, the entire visual field is colored in green making the viable cells hardly detectable.
  • the quenching dye solution is filtered through a filter with perforation of 0.2 ⁇ m to avoid contamination with disturbing microbes.
  • an antiseptic such as sodium azide can be contained as necessary.
  • the sodium azide for example, is added in an amount of final concentration of 0.1 to 5 mg/mL.
  • a quenching dye solution is dropped on a filtration surface of the filter or on an adhesion layer (a surface of capturing microbes) of the adhesion sheet, or alternatively, the filter or the adhesion sheet is dipped in a quenching dye solution, and left at 2 to 40°C for 1 to 10 sec and then excessive quenching dye solution is blown away using a blower.
  • the sample is mixed with a fluorescent dye solution and a quenching dye solution and stained. After filtration, the stained sample is transferred onto an adhesion sheet.
  • Excitation light for the fluorescent dye (with a wavelength in the range of 400 to 495 nm in the case of CFDA, for example) illuminates the filter or the adhesion sheet that has been stained with the fluorescent dye and the quenching dye.
  • An image of the fluorescence on the filtration surface of the filter or on the surface of adhesion layer of the adhesion sheet is saved using a CCD camera, a color camera, a monochromatic camera, or the like means.
  • an image of fluorescence is saved preferably through a filter that selectively transmit light with the wavelength of fluorescence of the fluorescent dye, to save a image of solely the fluorescence of the fluorescent dye emitted from the viable cells.
  • a filter that selectively transmits light with a wavelength in the range of 510 to 550 nm, but does not transmit light with a wavelength longer than 550 nm.
  • the fluorescence from the inclusions stained with the fluorescent dye is absorbed and quenched by the quenching dye and further, the fluorescence emitted by the quenching dye absorbing and consuming the energy of the fluorescence of the fluorescent dye is not saved. Therefore, the viable cells can be detected by bright spots of fluorescence emitted by the fluorescent dye in the viable cells.
  • the detection of the bright spots (or cells) can be carried out using a commercially available image analyzing software for example, "Optimas" (trade mark, a product of Media Cybernetics Corporation), though possible by visual observation.
  • Weak light emission that may cause noises in the counting can be eliminated by saving the fluorescent image through a neutral density filter, or more preferably, by an electronic procedure of image processing by setting a threshold value.
  • image processing can be conducted by the following procedure, for example.
  • a method of the invention also allows to save and form color images of both the fluorescence coming from the fluorescent dye and the fluorescence coming from the quenching dye.
  • the viable cells correspond to the bright spots of fluorescence coming from the fluorescent dye and the inclusions correspond to the bright spots of fluorescence coming from the quenching dye.
  • the latter fluorescence has a wavelength longer than that of the fluorescent dye.
  • the bright spots of fluorescence coming from the fluorescent dye are detected by visual observation or by using commercially available image analysis software as described above.
  • the weak light emission that may cause noises in the counting can be eliminated, as in the previous case, by saving the image of fluorescent light through a neutral density filter, or the saved image of fluorescent light can be electronically treated in the image processing by setting a threshold value.
  • the image of fluorescent light is preferably saved after enlarging the image using an optical element such as lens so that the size of the cells to be detected is equivalent to or larger than the size of the pixel of the image pick-up element.
  • the magnification can be appropriately selected depending on the size of the cells to be detected and generally a magnification from 10 to 1,000 is adequate.
  • the number of viable cells in the sample can be determined from the number of detected bright spots (, which correspond to viable cells,) as measured above, for example, according to a method of measuring total number of microbes described in "Guide to Health Control of Foods (Microorganisms edition)" (in Japanese; supervised by Bureau of Environmental Health, Ministry of Health and Welfare of the Japanese Government, and published by Japan Food Hygiene Association). Observing 16 fields or more, the total number (A) of bright spots (corresponding to viable cells) in the observed fields is determined.
  • C A ⁇ Sm / Sp ⁇ V , where V is a volume of the liquid sample used in the measurement, Sm is a surface area of filtration of the filter, and Sp is a total area of observed fields.
  • Fig. 2 shows a detection apparatus used in a preferred embodiment of a method of detecting viable cells according to the invention.
  • This detection apparatus 10 comprises a stage 2, a microscope tube 3, a lens 4, a band-pass filter 5, an image saving means 6, an excitation light source 7, a band-pass filter 8, and a dichroic mirror 9.
  • Sample 1 is fixed on the stage 2. (The sample 1 is fixed on a filter or transferred on an adhesive sheet, and stained with a fluorescent dye and a quenching dye.) An excitation light for the fluorescent dye is irradiated onto the sample 1 through an optical system for irradiating the excitation light consisting of the excitation light source 7, the band-pass filter 8, the microscope tube 3, the dichroic mirror 9, and the lens 4.
  • An image of the fluorescent light emitted from the sample 1 is saved in the image saving means 6 through an optical system for collecting fluorescence consisting of the lens 4, the dichroic mirror 9, the microscope tube 3, and the band-pass filter 5.
  • the image of the fluorescent light emitted from the sample 1 is enlarged by the lens 4, filtered by the band-pass filter 5, and saved in the image saving means 6.
  • the lens 4 enlarges the fluorescence image so that the size of the cells to be detected is equivalent to or larger than the size of the pixel of the image pick-up element.
  • the band-pass filter 5 transmits light with a wavelength of the fluorescent light emitted by the fluorescent dye, but does not transmit light with a wavelength of the light emitted by the quenching dye. Therefore, in this optical system for collecting fluorescence, the image saving means 6 saves solely the fluorescence of the fluorescent dye emitted from viable cells.
  • the image saving means can be a CCD camera, a color camera, or a monochromatic camera.
  • a color camera is used for an image saving means, and a color image is saved including both the fluorescence emitted by the fluorescent dye and the fluorescence emitted by the quenching dye.
  • the detection apparatus 10 can further comprise a means to detect the fluorescent light emitted by the fluorescent dye and to perform image processing on the fluorescence image saved in the image saving means 6, and a means to count the number of bright spots on the processed image.
  • a computer can be used for the means to perform image processing and for the means to count the number of bright spots.
  • Such a computer preferably installs the image processing program and the image analysis program as described in the process (3) above.
  • the light emitted by the excitation light source 7 passes through the band-pass filter 8 that transmits light with a wavelength of 400 nm to 495 nm, is reflected by a dichroic mirror 9 that reflects light with a wavelength shorter than 500 nm and transmits light with a wavelength longer than 500 nm, and illuminates the sample 1 by an excitation light of 400 nm to 495 nm.
  • the wavelength of the fluorescence of the CFDA overlaps the absorption wavelength of the Phenol Red and the absorption wavelength of the Cresol Red. Consequently, the Phenol Red and the Cresol Red absorb and quench the fluorescence of the CFDA, and the inclusions stained with the quenching dye emit red light in the range of 550 nm to 800 nm.
  • the viable cells are saved with the green light originated from CFDA and the inclusions other than the viable cells are saved with the red light originated from Phenol Red or Cresol Red.
  • the microtube was then turned upside down and the supernatant was discarded. After removing fat by swabbing with a sterile cotton tip stick, 100 ⁇ L of PBS was added to the microtube. Repeating suction and ejection using a pipette, suspension was formed of the precipitation, and then 1 mL of the PBS was added again to disperse the microbes.
  • the funnel part of the filtration equipment was dismounted and the membrane filter was detached.
  • An adhesive sheet (manufactured by Nitto Denko Corporation), which is not fluorescent and has a cellophane tape-like form, was adhered to the filtration surface of the membrane filter and the microbes and other substance on the membrane filter were transferred to the adhesion surface of the adhesive sheet.
  • the CFDA solution in an amount of 300 ⁇ L was dropped and spread on the adhesion surface of the adhesive sheet with the transferred microbes and other substance, and held stationary for 1 min at 25°C. Then the adhesive sheet was rinsed three times with 300 ⁇ L of cleaning liquid to wash excessive CFDA away.
  • stomach bag Ten grams of the solid food (namul of spinach) to which Escherichia coli (about 10 7 ) was added was aliquoted into a stomach bag.
  • the stomach bag is made of flexible plastic film such as polyethylene and sterilized by radiation. There is a compartment inside the stomach bag partitioned by non-woven fabric or a plastic film with perforation of about 0.28 mm diameter, and a liquid component can be extracted excluding relatively large solid bodies from the compartment.
  • the aliquot of the solid food is supplied with 90 mL of sterile phosphate buffer saline and, crushed and dispersed using a stomacher.
  • a stomacher is an apparatus for extracting live microbes contained in food to liquid. In the stomacher, the stomach bag containing food is placed between a fixed plate and a paddle and the food is crushed and dispersed by squeezing the bag containing food while moving the paddle back and forth.
  • the liquid portion was withdrawn from the stomach bag. Using the liquid, viable microbes were detected in a procedure similar to that in Example 1. The results are shown in Figs. 4 and 5 .
  • Fig. 4 shows the effectiveness in detecting viable microbes in various pH values in a fluorescent dye of CFDA solution and a quenching dye of Phenol Red solution. (The pH values were equal in both dye solutions.)
  • Fig. 5 is a fluorescent image of the adhesive sheet on which the microbes and other substances on the membrane filter were transferred, as saved by the fluorescent microscope without staining.
  • (b) is a fluorescence image of the adhesive sheet on which the microbes and other substances on the membrane filter were transferred, saved by the fluorescence microscope after staining with only a Phenol Red solution (pH7). Bright spots indicating viable microbes are not observed in Fig. 5(a) , while a large number of bright spots indicating viable microbes are observed in (b), showing penetration of the Phenol Red into the microbes.
  • the quenching dye of Phenol Red does not absorb and quench the fluorescence of the fluorescent dye in viable microbes, while it absorbs and quenches the fluorescence of the fluorescent dye coming from the inclusions other than the viable microbes.
  • a method of detecting viable cells of the invention can be utilized for detecting and quantifying viable cells in the field of medicines, agricultural chemicals, food health control, and in the research fields of medical science, pharmacy, and biology.

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Claims (12)

  1. Verfahren zum Nachweisen von lebensfähigen Zellen, umfassend die Schritte:
    (1) Fluoreszenzfärben von Zellen in einer Probe durch Zugeben eines Fluoreszenzfarbstoffs zu der Probe oder durch In-Kontakt-Bringen der Probe mit dem Fluoreszenzfarbstoff;
    (2) Zugeben eines quenchenden Farbstoffs, der in der Lage ist, die Fluoreszenz des Fluoreszenzfarbstoffs zu absorbieren, zu der Probe, die mit dem Fluoreszenzfarbstoff gefärbt ist, oder In-Kontakt-Bringen der Probe mit dem quenchenden Farbstoff; und
    (3) Beleuchten der Probe, die mit dem Fluoreszenzfarbstoff und dem quenchenden Farbstoff gefärbt ist, mit Anregungslicht für den Fluoreszenzfarbstoff, und Sammeln und Nachweisen von Fluoreszenzlicht, das von der Probe emittiert wird;
    wobei
    in Schritt (2), (a) der quenchende Farbstoff durch eine Membran einer lebensfähigen Zelle durchgängig ist, (b) der quenchende Farbstoff nicht die Fluoreszenz des Fluoreszenzfarbstoffs bei einem pH-Wert in den lebensfähigen Zellen absorbiert, und (c) der quenchende Farbstoff die Fluoreszenz des Fluoreszenzfarbstoffs bei einem pH-Wert absorbiert, der im Wesentlichen zu dem pH-Wert in den lebensfähigen Zellen unterschiedlich ist, und der quenchende Farbstoff zu der Probe zugegeben oder die Probe mit dem quenchenden Farbstoff bei einem pH-Wert in Kontakt gebracht wird, der zu dem pH-Wert in den lebensfähigen Zellen im Wesentlichen unterschiedlich ist; und
    in Schritt (3) die Probe, die mit dem Fluoreszenzfarbstoff und dem quenchenden Farbstoff gefärbt ist, bei einem pH-Wert gehalten wird, der zu dem pH-Wert in den lebensfähigen Zellen unterschiedlich ist.
  2. Verfahren zum Nachweisen von lebensfähigen Zellen gemäß Anspruch 1, wobei der Fluoreszenzfarbstoff in der Lage ist, nur lebensfähige Zellen zu färben.
  3. Verfahren zum Nachweisen von lebensfähigen Zellen nach Anspruch 1 oder Anspruch 2, wobei der Fluoreszenzfarbstoff eine Substanz ist, die die Nukleinsäure markiert, um sie fluoreszierend zu machen, oder ein Enzymsubstrat, das bei enzymatischen Abbau fluoreszierend wird.
  4. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1-3, wobei der quenchende Farbstoff eine Verbindung mit einer konjugierten Doppelbindung(en) ist, die Licht mit einer Wellenlänge der Fluoreszenz des Fluoreszenzfarbstoffs absorbiert.
  5. Verfahren zum Nachweisen von lebensfähigen Zellen nach Anspruch 4, wobei der quenchende Farbstoff eine aromatische Verbindung mit wenigstens 2 aromatischen Ringen ist.
  6. Verfahren zum Nachweisen von lebensfähigen Zellen nach Anspruch 4, wobei der quenchende Farbstoff eine aromatische Verbindung mit wenigstens einem kondensierten aromatischen Ring ist.
  7. Verfahren zum Nachweisen von lebensfähigen Zellen nach Anspruch 4, wobei der quenchende Farbstoff eine Verbindung mit einer ungesättigten Kohlenwasserstoffstruktur ist.
  8. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1-4, wobei der quenchende Farbstoff ausgewählt ist aus Verbindungen, dargestellt durch die Formeln (I) bis (IX), und Anthocyaninen,
    Figure imgb0033
    Figure imgb0034
    Figure imgb0035
    Figure imgb0036
    Figure imgb0037
    Figure imgb0038
     wobei in den Formeln (I), (II), (III) und (V) jedes von R1 bis R11, die dieselben oder unterschiedlich sein können, ein Wasserstoffatom, eine Methylgruppe, eine aliphatische Kette oder einen Ester einer Fettsäure von zwei oder mehr Kohlenstoffatomen, Jod oder Brom darstellt,
    Figure imgb0039
     wobei in der Strukturformel (VII) Me Eisen, Kupfer oder Magnesium darstellt; jedes von R1 bis R7 eine Kohlenwasserstoffgruppe darstellt; und jedes von R8 bis R12 eine Kohlenwasserstoffgruppe, ein Wasserstoffatom oder ein Kohlenstoffatom darstellt,
    Figure imgb0040
     wobei in der Strukturformel (VIII) jedes von R1 bis R8 eine Kohlenwasserstoffgruppe darstellt; jedes von R9 bis R12 eine Kohlenwasserstoffgruppe, ein Wasserstoffatom oder ein Kohlenstoffatom darstellt,
    Figure imgb0041
     wobei in der Strukturformel (IX) jedes von R1 bis R4 eine Kohlenwasserstoffgruppe, ein Wasserstoffatom oder ein Kohlenstoffatom darstellt, und n eine ganze Zahl von 1 bis 11 ist.
  9. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1 bis 8, wobei der Schritt (2) einen quenchenen Farbstoff verwendet, der die Fluoreszenz des Fluoreszenzfarbstoffs bei einem alkalischen pH-Wert absorbiert, und der Schritt (3) durchgeführt wird, wobei die mit dem Fluoreszenzfarbstoff und dem quenchenden Farbstoff gefärbte Probe bei einem alkalischen pH-Wert gehalten wird.
  10. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1-8, wobei der Schritt (2) einen quenchenden Farbstoff verwendet, der die Fluoreszenz des Fluoreszenzfarbstoffs bei einem sauren pH-Wert absorbiert, und der Schritt (3) durchgeführt wird, wobei die mit dem Fluoreszenzfarbstoff und dem quenchenden Farbstoff gefärbte Probe bei einem sauren pH-Wert gehalten wird.
  11. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1-10, wobei in Schritt (3) nur Licht mit einer Wellenlänge der Fluoreszenz des Fluoreszenzfarbstoffs gesammelt wird, die von der Probe emittiert wird, und wobei fluoreszierendes Licht als ein Bild gespeichert wird.
  12. Verfahren zum Nachweisen von lebensfähigen Zellen nach einem der Ansprüche 1-10, wobei in Schritt (3) die von der Probe emittierte Fluoreszenz gesammelt und als ein Bild in Farbe gespeichert wird, und wobei die Fluoreszenz, die aus dem Fluoreszenzfarbstoff stammt, von der Fluoreszenz unterschieden wird, die aus anderen Quellen stammt.
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